Ischemic preconditioning (IPC) has proved to be one of the most effective methods of reducing ischemic brain damage in animal models of stroke. Attention has focused on the mechanisms by which neurons may be protected by such preconditioning. However, we have found that IPC also protects the endothelium, which forms the blood-brain barrier, from ischemic damage. 'Ischemic' preconditioning also protects cerebral endothelial cells in vitro from oxygen glucose deprivation induced injury indicating that there can be direct preconditioning of the endothelium. Damage to the cerebral endothelium may potentiate ischemic brain injury in a number of ways and determining mechanisms to reduce endothelial damage is particularly pertinent at the moment considering the role of endothelial injury (hemorrhagic transformation) in limiting the use of tissue plasminogen activator for the treatment of ischemic stroke. We will examine the mechanisms involved in cerebral endothelial preconditioning both in vivo (rat middle cerebral artery occlusion) and in vitro (primary cultures of rat cerebral microvessel endothelial cells). The in vitro experiments will facilitate exploration of the mechanisms involved in preconditioning while assuring that the preconditioning acts directly on the endothelium. The in vivo experiments will ensure that the mechanisms elucidated in vitro also occur in the whole animal as well as allowing an assessment of the effects of preconditioning on other parameters (such as blood flow, capillary morphology and infarction). Overall the proposal has three main goals. 1) Determine the time course of preconditioning and the extent of its effects on the endothelium (Specific Aim 1). 2) Determine the events that can trigger endothelial preconditioning (Specific Aim 2). 3) Determine what mechanisms are triggered to protect the endothelium (Specific Aim 3). These experiments should provide information on: A) Endogenous defense mechanisms that protect the cerebral endothelium from ischemic injury and which may be therapeutic targets. B) The role of endothelial preconditioning in the effects of IPC on the brain. C) The role of endothelial injury in the overall effects of ischemia on the brain.